NeuroD, a basic helix-loop-helix (bHLH) transcription factor, is required for islet formation and beta cell survival. We propose to determine the cellular basis for failure to form islets in NeuroD-null mice. Although NeuroD affects beta cells more than other cell types, loss of beta cells alone in other gene targeted mutant mice (e.g., PAX-4) does not result in failure to form islets. Thus, our mice provide an excellent opportunity to determine which cell types control islet biogenesis and whether islet formation has a direct role in maturation of beta cells. Here, we hypothesis that NeuroD affects development of cells other than beta cells, which are required for islet formation. As a first step to determine the role of NeuroD in generation of different cell types in the pancreas, we will perform a cell lineages analysis of NeuroAD expressing cells. Once we identify the non-beta cells that are of NeuroD-lineage, future experiments will include reintroduction of NeuroD into these cell types to restore islet formation. We also propose that NeuroD AND Nkx2.2, another essential pancreatic regulatory factor, are required for beta cell maintenance and regeneration in the adult islet. Nkx2.2 is a homeobox transcription factor that is required for the development and differentiation of three of the four endocrine cell types in embryonic mice: a deletion of Nkx2.2 adversely affects the development of alpha, beta and PP cells. NeuroD and Nkx2.2 are both high expressed in adult pancreatic beta cells, raising the possibility that they might also play roles in mature beta cells. In fact, while many NeuroD-null cells in the brain die during development, some die after morphological differentiation occurs, suggesting that NeuroD is required for maintenance of these neurons. Furthermore, several Nkx2 family members have been shown to play a role in cell-type maintenance in the tissues where they are expressed post-natally (Schott et al 1998; Shiratori et al 2001). Based on these observations and the expression patterns of both genes in mature beta cells, we hypothesize that NeuroD and Nkx2.2 are required for the maintenance and possibly regeneration of beta cells. To test this hypothesis, we propose to generate mice from which the neuroD or the Nkx2.2 allele will be conditionally eliminated in adult beta cells. Specific Aim 1. Identify the cell types generated from NeuroD expressing cells. Specific Aim 2. Determine whether NeuroD/Beta2 and Nkx2.2 are required for mature beta cell function and maintenance. Specific Aim 3. Determine whether NeuroD and Nkx2.2 are required for beta cell regeneration.